Attaching substances to micro-organisms

ABSTRACT

The invention relates to surface display of proteins on micro-organisms via the targeting and anchoring of heterologous proteins to the outer surface of cells such as yeast, fungi, mammalian and plant cells, and bacteria. The invention provides a proteinaceous substance comprising a reactive group and at least one attaching peptide which comprises a stretch of amino acids having a sequence corresponding to at least a part of the consensus amino acid sequence listed in FIG.  10  and comprises a method for attaching a proteinaceous substance to the cell wall of a micro-organism comprising the use of said attaching peptide.

Heterologous surface display of proteins (Stahl and Uhlen, TIBTECH May1997, 15±185–192) on recombinant micro-organisms via the targeting andanchoring of heterologous proteins to the outer surface of host-cellssuch as yeast, fungi, mammalian and plant cells, and bacteria has beenpossible for several years. Display of heterologous proteins at thesurface of these cells has taken many forms, varying from the expressionof reactive groups such as antigenic determinants, heterologous enzymes,(single-chain) antibodies, polyhistidyl tags, peptides, and othercompounds. Heterologous surface display has been applied as a tool forapplied and fundamental research in microbiology, molecular biology,vaccinology and biotechnology, and several patent applications have beenfiled.

Yet another application of bacterial surface display has been thedevelopment of live-bacterial-vaccine delivery systems. The cell-surfacedisplay of heterologous antigenic determinants has been consideredadvantageous for the induction of antigen-specific immune responses whenusing live recombinant cells for immunisation. Another application hasbeen the use of bacterial surface display in generating whole-cellbioadsorbents or biofilters for environmental purposes,microbiocatalysts, and diagnostic tools.

In general, one has used chimeric proteins consisting of an anchoring ortargeting part specific and selective for the recombinant organism usedand has combined this part with a part comprising a reactive group asdescribed above. A well known anchoring part for example comprise theso-called LPXTG box, that binds covalently to a Staphylococcus bacterialsurface, i.e. in the form of a fully integrated membrane protein. Inthis way, chimeric proteins are composed of at least two (poly)peptidesof different genetic origin joined by a normal peptide bond. Forexample, in patent application WO 94/18830 relating to the isolation ofcompounds from complex mixtures and the preparation of immobilisedligands (bioadsorbents), a method has been claimed for obtaining such aligand which comprises anchoring a binding protein in or at the exteriorof the cell wall of a recombinant cell. Said binding protein isessentially a chimeric-protein produced by said recombinant cell, and iscomposed of an N-terminal part, derived from for example an antibody,that is capable of binding to a specific compound joined with aC-terminal anchoring part, derived from an anchoring protein purposelyselected for being functional in the specific cell chosen. In patentapplication WO 97/08553 a method has been claimed for the targeting ofproteins selectively to the cell wall of Staphylococcus spp, using asanchoring proteins long stretches of at least 80–90 amino acid longamino acid cell wall-targeting signals derived from the lysostaphin geneor amidase gene of Staphylococcus which encode for proteins thatselectively bind to Staphylococcus cell wall components.

Vaccine delivery or immunisation via attenuated bacterial vector strainsexpressing distinct antigenic determinants against a wide variety ofdiseases is now commonly being developed. Recently, mucosal (for examplenasal or oral) vaccination using such vectors has received a great dealof attention. For example, both systemic and mucosal antibody responsesagainst an antigenic determinant of the hornet venom were detected inmice orally colonised with a genetically engineered human oral commensalStreptococcus gordonii expressing said antigenic determinant on itssurface (Medaglini et al., PNAS 1995, 2; 6868–6872). Also, a protectiveimmune response could be elicited by oral delivery of a recombinantbacterial vaccine wherein tetanus toxin fragment C was expressedconstitutively in Lactococcus lactis (Robinson et al., NatureBiotechnology 1997, 15; 653–657). Especially mucosal immunisation as ameans of inducing IgG and secretory IgA antibodies directed againstspecific pathogens of mucosal surfaces is considered an effective routeof vaccination. Immunogens expressed by bacterial vectors are presentedin particulate form to the antigen-presenting cells (for exampleM-cells) of the immune system and should therefore be less likely toinduce tolerance than soluble antigens. In addition, the existence of acommon mucosal immune system permits immunisation on one specificmucosal surface to induce secretion of antigen-specific IgA, and otherspecific immune responses at distant mucosal sites. A drawback to thisapproach is the potential of the bacterial strain to cause inflammationand disease in itself, potentially leading to fever and bacteraemia. Analternative approach avoids the use of attenuated bacterial strains thatmay become pathogenic themselves by choosing recombinant commensalbacteria as vaccine carriers, such as Streptococcus spp. and Lactococcusspp.

However, a potential problem with such recombinant organisms is thatthey may colonise the mucosal surfaces thereby generating a long termexposure to the target antigens expressed and released by theserecombinant micro-organisms. Such long term exposure can cause immunetolerance. In addition, the mere fact alone that such organisms aregenetically modified and contain recombinant nucleic acid is meetingconsiderable opposition from the (lay) public as a whole, stemming froma low level of general acceptance for products containing recombinantDNA or RNA. Similar objections exist against the use of (evenattenuated) strains of a pathogenic nature or against proteins or partsof proteins derived from pathogenic strains. However, as explainedabove, present techniques of heterologous surface display of proteins ingeneral entail the use of anchoring or targeting proteins that arespecific and selective for a limited set of micro-organisms which ingeneral are of recombinant or pathogenic nature, thereby greatlyrestricting-their potential applications.

The invention provides substances and methods to anchor or attach saidsubstances to a cell-wall or cell-wall component of a wide range ofmicro-organisms. A preferred embodiment of the invention providessubstances and methods to attach said substances to non-recombinantmicro-organisms. Said substances provided by the invention are notlimited to (chimeric) proteins alone, but can be fully or only partly ofa peptide nature, whereby a peptide part is (covalently) joined to anon-peptide moiety. The invention provides a proteinaceous substancecomprising at least one stretch of amino acids derived from a firstmicro-organism which substance is capable of attaching to a cell-wall ofa second micro-organism. Said substance according to the invention isfor example produced by a first micro-organism (for example amicro-organism from which the knowledge about the sequence of saidstretch of amino acids originates, but another (recombinant)micro-organism can produce said substance as well). After its productionsaid substance is harvested, optionally stored for future use, and thenbrought in contact with said second micro-organism, where it attaches toits cell-wall. Alternatively, said substance is produced synthetically,by using established peptide synthesis technology. A preferredembodiment of the invention provides a substance wherein said secondmicro-organism is a non-recombinant micro-organism. With a substanceprovided by the invention it is now possible to attach or anchor for aexample a heterologous or chimeric protein produced by a recombinantmicro-organism to an innocuous non-recombinant micro-organism.

A preferred embodiment of the invention provides a proteinaceoussubstance wherein said stretch of amino acids has a sequencecorresponding to a consensus sequence listed in FIG. 10 (SEQ ID NOS.17), or wherein said stretch of amino acids (herein also calledattaching peptide) has a sequence corresponding to a sequence selectedfrom those listed in FIG. 11 (SEQ ID NOS. 20–110), or a homologoussequence derived from another species. The sequences listed in FIG. 11(SEQ ID NOS. 20–110), and sequences homologuous thereto, are found in avariety of species, both micro-organisms and higher organisms, anexample of such a higher organism is C. elegans. Preferably, theattaching peptide is derived from any one of the proteins listed in FIG.11 (SEQ ID NOS. 20–110), more preferably said attaching peptidecomprises an amino acid sequence as shown in FIG. 10 (SEQ ID NOS.14–17), or a sequence derived thereof. For example, the inventionprovides a proteinaceous substance wherein said attaching peptide isderived from the major peptidoglycan hydrolase of Lactococcus lactis.(SEQ ID NOS. 14–16 and 20–22).

Yet another preferred embodiment of, the invention provides aproteinaceous substance wherein said second microorganism is selectedfrom any of the group of Gram-positive bacteria and Gram-negativebacteria. Examples are microorganisms, such as Bacillus subtilis (SEQ IDNOS. 75–78, 81–87, 104, 107, 109–110); Clostridium beij erinckii,Lactobacillus plantarum, Lb. buchneri, Listeria inocua, Streptococcusthermophilus, Enterococcus faecalis (SEQ ID NOS. 23–27), E. coli (SEQ IDNOS. 64, 66, 72–73, 79, 106), and others.

The invention provides a proteinaceous substance which is additionallycomprising a reactive group. For example, the invention provides aproteinaceous substance comprising a reactive group such as an antigenicdeterminant, heterologous enzyme, (single-chain) antibody or fragmentthereof, polyhistidyl tag, fluorescing protein, luciferase, bindingprotein or peptide, or another substance such as an antibiotic, hormone,non-peptide antigenic determinant, carbohydrate, fatty acid, aromaticsubstance and reporter molecule, and an anchoring or targeting proteinor part thereof (herein also called attaching peptide) useful inheterologous surface display which is both broadly reactive with cellwall components of a broad range of micro-organisms.

For example, the invention provides a substance wherein said reactivegroup is a non-protein moiety, for example is selected from the group ofantibiotics, hormones, aromatic substances and reporter molecules. Saidsubstance is constructed by binding for example an antibiotic, such aspenicillin or tetracycline, but various other antibiotics can be used,or a hormone, such as a steroid hormone, or any other compound to anattaching peptide provided by the invention. Such binding can beachieved by various techniques known in the art, and thereby can labelor “flag” the attaching peptide. A preferred example is the binding ofan attaching peptide to a reporter molecule such as FITC, or HRPO,whereby tools are generated that can be used in diagnostic assay wherebymicro-organisms having peptidoglycan are detected. Similarly, anattaching peptide with an antibiotic bound thereto can be used in vivoby for example parenteral administration into the bloodstream of humansor animals or in vitro to bind to such micro-organisms havingpeptidoglycan, thereby increasing the concentration of antibiotic aroundsaid organism, which then gets killed by the antibiotic action.

The invention provides a substance wherein said reactive group is aprotein moiety, for example selected from the group of antigenicdeterminants, enzymes, (single-chain) antibodies or fragments thereof,polyhistidyl tags, fluorescing proteins, binding proteins or peptides.For example, the invention provides a protein, which comprises asreactive group a protein or (poly)peptide. Also, the invention providesa nucleic acid molecule encoding a protein provided by the invention.Such a nucleic acid molecule (being single- or double stranded DNA, RNAor DNA/RNA) at least comprises nucleic acid sequences specificallyencoding a attaching peptide as well as nucleic acid sequencesspecifically encoding the reactive group polypeptide, but canadditionally also comprise other nucleic acid sequences, which forexample encode a signal peptide, or comprise for example promoter and/orregulatory nucleic acid sequences. The invention also provides a vectorcomprising a nucleic acid molecule encoding a protein provided by theinvention.

The invention provides a proteinaceous substance comprising a reactivegroup joined with or bound to at least one attaching peptide whichcomprises a stretch of amino acids corresponding to the consensus aminoacid sequence listed in FIG. 10, said substance capable of attaching oranchoring or binding to a cell wall component of a micro-organism.

Corresponding to is defined as having an amino acid sequence homologousto the consensus amino acid sequence listed in FIG. 10, or having anamino acid sequence derived of the sequence listed in FIG. 10 whichderived sequence comprises a functionally equivalent stretch of aminoacids.

Preferably, the attaching peptide is derived from any one of theproteins listed in FIG. 11, or a protein having a repeat sequencerelated or homologous to the sequence listed in FIG. 10, more preferablysaid attaching peptide comprises an amino acid sequence as shown in FIG.10, or a sequence derived thereof. Homology between the various aminoacid sequences of related attaching peptides provided by the inventioncan for instance be determined by performing a homology search betweenamino acid sequences, such as for example can be found in a proteindatabase, such as the SWISSPROT, PIR and Genbank databases, using acomputer programme, such as the BLAST programme, that can determinehomology between amino acid sequences. For example, the inventionprovides a proteinaceous substance wherein said attaching peptide isderived from the major peptidoglycan hydrolase of Lactococcus lactis.The invention provides a proteinaceous substance comprising a reactivecompound wherein at least two stretches of amino acids, corresponding toan attaching peptide sequence, are located adjacent to each other,possibly separated by one or more amino acid residues. Said stretches orrepeats can be separated by a short distance, for example 3–6 to 10–15amino acids apart, or by a medium distance 15–100 amino acids apart, orby longer distances (>100 amino acid residues apart). Examples of suchdistances can be found in FIG. 11, but longer distances are alsopossible. The distances between said stretches or repeats can also beused for an (additional) reactive group, whereby a reactive group isinserted between repeats, thereby allowing an even better anchoring to acell wall component. A preferred embodiment provided by the invention isa proteinaceous substance comprising a reactive group and at least oneattaching peptide which comprises a stretch of amino acids having asequence corresponding to the consensus amino acid sequence listed inFIG. 10, wherein said substance is capable of attaching to a cell wallcomponent of a micro-organism, such as can be found among from any ofthe group of yeast, moulds, Gram-positive bacteria and Gram-negativebacteria. Examples are micro-organisms, such as Bacillus subtilis,Clostridium beijerinckii, Lactobacillus plantarum, Lb. buchneri,Listeria inocua, Streptococcus thermophilus, Enterococcus faecalis, E.coli, and others. A preferred embodiment provided by the invention is aproteinaceous substance which is capable of attaching to a cell wallcomponent of a conventional (non-recombinant) micro-organism. In thisembodiment, the invention provides for example non-recombinant organismswhich displaying heterologous proteins, these may colonise the mucosalsurfaces without causing problems such as immune tolerance, since theydo not generate a long term exposure to the target antigens expressed.In addition, the mere fact alone that such organisms provided by theinvention are not genetically modified and do not contain recombinantnucleic acid will alleviate the opposition from the (lay) public as awhole against recombinant micro-organisms, which is stemming from a lowlevel of general acceptance for products containing recombinant DNA orRNA. Similar objections that exist against the use of (even attenuated)strains of a pathogenic nature or against proteins or parts of proteinsderived from pathogenic strains are now also overcome by the invention,in that is now possible to attach a proteinaceous substance to anon-recombinant, non-pathogenic micro-organism, such as L. lactis whichis generally considered as safe. The invention provides a proteinaceoussubstance comprising a reactive group such as an antigenic determinant,(heterologous) enzyme, (single-chain) antibody or fragment thereof,polyhistidyl tag, fluorescing protein, luciferase, binding protein orpeptide, or another compound such as an antibiotic, hormone, non-peptideantigenic determinant, carbohydrate, fatty acid, aromatic compound andreporter molecule, and an anchoring or targeting protein or part thereof(herein also called attaching peptide) useful in heterologous surfacedisplay which is both broadly reactive with cell wall components of abroad range of micro-organisms. Said attaching peptide is preferablyderived from a micro-organism which is generally recognised as safe(G.R.A.S.), thereby greatly enhancing the potential of applications ofthe heterologous surface display technique. Lactococcus lactis is anon-pathogenic, non-invasive, and non-colonising Gram-positive bacteriumwhich is not adapted for growth in body or even the gut; it does notbelong to the commensal species of lactic acid bacteria. L. lactis has ahistory of safe use of several thousand years. The major cell wallhydrolase AcmA of the Gram-positive bacterium Lactococcus lactis subsp.cremoris MG1363 is an N-acetylmuramidase which is required for cellseparation and is responsible for cell lysis during stationary phase.The protein consists of three separate domains (FIG. 9, Buist et al., J.Bacteriol. (1995) 177:1554–1563) of which the first 57 amino acids ofthe N-terminal domain encompasses the signal peptide needed forsecretion. This domain is followed by the active site domain runningfrom the Ala at position 58 to Ser-218. The active site domain wasoverproduced in and purified from Escherichia coli as a thioredoxinfusion protein. The AcmA part was released by proteolytic cleavage withenterokinase and shown to be active in vitro. Three homologous repeatedregions (or stretches of amino acids) of 35–55, more often 40–50 aminoacid residues are present in the C-terminus of for example AcmA whichare separated by non-homologous sequences (FIG. 10). The repeatsequences of AcmA (cA) can be deleted and additional repeat sequencescould be added without impairing cell wall hydrolysing activity invitro. The AcmA deletion derivatives lacking one or two repeat sequencesand the protein containing at least one additional repeat were able tobind to lactococcal cells when added from the outside. The derivativelacking all three repeats did not bind to the cells nor did the purifiedactive site domain. The invention provides an attaching peptide thatcomprise at least one repeat sequence as shown in FIG. 10 or a sequencethat is similar to the sequence of FIG. 10, similar being defined ascomprising at least a part of a consensus sequence as shown in FIG. 11.Also, attaching peptides are provided by the invention which arecomprising amino acid sequences that are derived from a sequence asshown in FIG. 11. Derived herein meaning among others by comparison withheterologous sequences whereby a consensus sequence is obtained, orderived via conventional amino acid substitutions whereby amino acidsare being substituted by like amino acids, or derived via substitutionswhereby functional amino acids are being replaced by functionally alikeor better amino acids identified by methods such as PEPSCAN techniquesor replacement mapping. The invention provides a proteinaceous substancecomprising a reactive group and at least one attaching peptide whichcomprises a stretch of amino acids having a sequence corresponding to atleast a part of the consensus amino acid sequence provided in FIG. 10.Repeats similar to those in AcmA were for example shown to be present invarious cell wall hydrolases and other (secreted) proteins ofGram-positive and Gram-negative bacteria and other micro-organisms andconstitute a general cell wall binding domain in these proteins. Anattaching peptide comprising at least one AcmA repeat or an amino acidsequence similar to the AcmA repeat provided by the invention representsa general and broadly reactive tool to bind or attach reactive groupssuch as antigenic determinants, enzymes, antibodies, proteins orpeptides to cell walls of micro-organisms. Said repeat comprises apeptide composed of a stretch of amino acids having a sequencecorresponding to at least a part of the consensus amino acid sequenceprovided in FIG. 10. Furthermore, we also demonstrated that an attachingpeptide provided by the invention bound or attached to cells of other,e.g. non-recombinant micro-organisms, such as Bacillus subtilis,Clostridium beijerinckii, Lactobacillus plantarum, Lb. buchneri,Listeria inocua, Streptococcus thermophilus, Enterococcus faecalis, E.coli, and others. Binding of the attaching peptide and reactive groupjoined therewith, as provided by the invention is stable at pH valuesranging from 2–10. Moreover, the attaching peptide provided by theinvention is, when attached to the cell wall, protected againstproteolytic degradation. One embodiment of the invention is a proteinwherein the attaching peptide is derived from any of the proteins listedin FIG. 11. An example of such an attaching peptide is provided in theexperimental part of this description wherein an attaching peptidehaving a sequence as shown in FIG. 10, or a sequence similar thereto, isused. Furthermore, the invention provides a protein, which comprises asreactive group a protein or (poly)peptide. Also, the invention providesa nucleic acid molecule encoding a protein provided by the invention.Such a nucleic acid molecule (being single- or double stranded DNA, RNAor DNA/RNA) at least comprises nucleic acid sequences specificallyencoding a attaching peptide as well as nucleic acid sequencesspecifically encoding the reactive group polypeptide, but canadditionally also comprise other nucleic acid sequences, which forexample encode a signal peptide, or comprise for example promoter and/orregulatory nucleic-acid sequences. The invention also provides a vectorcomprising a nucleic acid molecule encoding a protein provided by theinvention. Such a vector can for example be a plasmid, phage, or virus,and can now be constructed using a nucleic acid provided by theinvention and routine skills of the art. Examples of such a vector canbe found in the experimental part of the description, other examples cane.g. be a baculovirus vector, or comparable vector viruses through whicha protein provided by the invention can be expressed or produced in(insect) cells. The invention also provides a host cell or expressionsystem comprising a nucleic acid molecule according to the invention ora vector according to the invention. Such a host cell expressing aprotein is in it self provided by the invention as a micro-organism towhich a protein provided by the invention is attached. Such a host cellor expression system can for example be a Gram-positive- orGram-negative bacterium, or a yeast cell or insect cell or plant- ormammalian cell, or even a cell-free expression system such as areticulocyte lysate, and can now be constructed or obtained using anucleic acid or vector provided by the invention and routine skills ofthe art. Examples of such a host cell or expression system can be foundin the experimental part of the description, other examples can beobtained using a nucleic acid or vector provided by the invention androutine skills of the art.

The invention provides a method for attaching a substance to the cellwall of a micro-organism comprising the use of an attaching peptidewhich comprises a stretch of amino acids having a sequence correspondingto at least a part of the consensus amino acid sequence provided in FIG.10. An example of the method provided by the invention is anchoring ofrecombinant poly(peptides), being (chimeric) proteins fused to the cellwall anchoring repeats of AcmA of Lactococcus lactis MG1363, to the cellwall of (Gram-positive) bacteria. The recombinant proteins are obtainedby the expression of DNA sequences encoding these recombinant(poly)peptides in a suitable production strain (e.g. E. coli or L.lactis) and subsequent purification of the expression products. Therecombinant proteins are than mixed, either in vitro or in vivo, with anon-recombinant target bacterium to obtain binding to the cell wall.Another example of the method provided by the invention is anchoring ofrecombinant poly(peptides), being (chimeric) proteins fused to the cellwall anchoring repeats of AcmA of Lactococcus lactis, to the cell wallof said recombinant Lactococcus lactis which produces the proteinitself. In a preferred embodiment of the method provided by theinvention the binding of (purified) proteins to bacterial cells uponaddition from the outside, the method is an excellent tool to anchorrecombinant proteins or other substances to non-recombinant bacterialcells.

A preferred method according to the invention comprises the use of anattaching peptide which is derived from the major peptidoglycanhydrolase of Lactococcus lactis. Another method according to theinvention is provided wherein said substance is a (poly)peptide or aprotein, for example being part of a protein provided by the invention.Post-translational modifications occurring to such a (poly)peptide orprotein are inherent to the host cell or expression system used, apost-translationally modified protein as provided by the invention istherefore also provided. However, yet another method according to theinvention is provided wherein said compound is selected from the groupcomposed of antibiotics, hormones, antigenic determinants, carbohydratechains, fatty acids, aromatic compounds and reporter molecules. Saidsubstance is constructed by binding for example an antibiotic, such aspenicillin or tetracycline, but various other antibiotics can be used,or a hormone, such as a steroid hormone, or any other compound to anattaching peptide provided by the invention. Such binding can beachieved by various techniques known in the art, and thereby can labelor “flag” the attaching peptide. A preferred example is the binding ofan attaching peptide to a reporter molecule such as FITC, or HRPO,whereby tools are generated that can be used in diagnostic assay wherebymicro-organisms having peptidoglycan are detected. Similarly, anattaching peptide with an antibiotic bound thereto can be used in vivoby for example parenteral administration into the bloodstream of humansor animals or in vitro to bind to such micro-organisms havingpeptidoglycan, thereby increasing the concentration of antibiotic aroundsaid organism, which than can get killed by the antibiotic action. Saidmicro-organism is preferably selected from any of the group of yeast,moulds, Gram-positive bacteria and Gram-negative bacteria. For example,the experimental part of this description describes mixing ofβ-lactamase::cA fusion protein with lactococcal cells which resulted inbinding to the cells whereas this was not the case when matureβ-lactamase not joined with an attachement protein was added. Also,fusion of β-lactamase of E. coli and α-amylase of Bacillus licheniformisto the attaching peptide provided by the invention and subsequentproduction of these fusion proteins resulted in active, secretedproteins which were located (attached) in L. lactis cell walls. Bindingof AcmA and the β-lactamase::cA fusion protein was also demonstrated toisolated lactococcal cell walls and SDS-washed cell walls (the majorpart of this fraction is peptidoglycan).

Anchoring of recombinant proteins to non-recombinant micro-organismssuch as lactococci (or other bacteria) or fungi, is especiallyattractive if the use of recombinant bacteria is not desired, e.g. infood processes or as pharmaceuticals for medical use such as in vaccinesor in anti-bacterial therapy. The invention provides for example vaccinedelivery or immunisation via micro-organisms provided by the inventionwhich are labelled with distinct antigenic determinants, which may bedirected against a wide variety of diseases. A protective immuneresponse can for example be elicited by oral delivery of a bacterialvaccine provided by the invention wherein tetanus toxin fragment C isattached via a protein provided by the invention to a non-recombinantLactococcus lactis. Such immunogens expressed by micro-organismsprovided by the invention are presented in particulate form to theantigen-presenting cells (for example M-cells) of the immune system andare therefore less likely to induce tolerance than soluble antigens. Inaddition, the existence of a common mucosal immune system permitsimmunisation on one specific mucosal surface to induce secretion ofantigen-specific IgA, and other specific immune responses at distantmucosal sites. The invention solves the drawback of earlier bacterialvaccines whereby the potential to flourish on mucosal surfaces of the(attenuated or recombinant) bacterial strain used can cause problemssuch as inflammation and disease in itself, potentially leading to feverand bacteraemia, or to the induction of immune tolerance. Also, theinvention avoids the potential risks that are involved when usingrecombinant DNA containing bacterial vectors for vaccination. In yetanother possible vaccine and vaccine use provided by the invention,certain (killed) micro-organisms with adjuvant properties (such as themycobacteria used in BCG) are labelled or loaded with a protein orsubstance composed of an antigenic determinant and an attaching peptide.These micro-organisms than function as adjuvant, thereby greatlyenhancing the immune response directed against the specific antigenicdeterminant. Yet another use provided by the invention comprisesanchoring proteins from the outside to a micro-organism which provides ameans to present proteins or peptides which normally can not beoverexpressed (and/or secreted) by said micro-organism. For example, thesubunit B of cholera toxin (CTB) can be overproduced in E. coli butexpression in L. lactis has been unsuccessful until now. The adjuvantactivity of CTB in experimental recombinant vaccines is well documentedand the ability of CTB or part thereof to bind to GM1 ganglioside oneucaryotic cell surfaces is of interest with respect to the use of L.lactis (or other Gram-positives) in vaccines which specifically requiretargeting to mucosal surfaces. Yet another medical use provided by theinvention is the addition of (purified) antigen::cA fusion proteins invivo by parenteral administration into the bloodstream of humans oranimals to combat bacterial infections. In this case the antigen::cAfusion protein is used as a “flag” for the immune system. The antigenicdeterminant of a protein provided by the invention being a subunit of avaccine regularly used for the immunisation of humans (preferablychildren) or animals, e.g. a subunit of the Rubella, Pertusis,Poliomyelitis, tetanus or measles vaccine. After delivery in thebloodstream, the “flag” will bind through the AcmA repeats to thepathogenic bacterium present in the blood. A “flag” protein provided bythe invention will then activate a memory response, i.e. the response tothe antigenic determinant present in the protein. The antibodies thusproduced recognise the “flag”—labelled bacteria, which will then beneutralised by the immune system. In this way the protein is used tostimulate a pre-existing (memory) immune response, non-related to thebacterial infection, to clear bacterial infections from the system. Yetanother use (which alternatively may be considered medical use or fooduse) provided by the invention is the use wherein a protein provided bythe invention has the ability to bind to cells, such as mucosal cells,e.g. of the gut. The reactive group of such a protein is in such a casefor example partly or wholly derived from a fimbriae protein or anothergut attachment protein, as for example present in various E. colistrains. Micro-organisms to which such a protein is attached willspecifically home or bind to certain areas of the gut, a property whichfor example is beneficial for certain bacterial strains (i.e.lactococcal or lactobacillar strains) used as a probiotic. In anotherfood or use of food provided by the invention, the protein or substanceprovided by the invention is a composed of a food additive (such as anenzyme or flavour compound) which affects quality, flavour, shelf-life,food value or texture, joined with an attaching peptide, andsubsequently attached or anchored to a micro-organism which is thanmixed with the foodstuff. The anchoring of such proteins to a bacterialcarrier offers the additional advantage that the additive can betargeted to a solid (bacteria-containing) matrix (e.g. curd) in aprocess for the preparation of food, e.g. cheese or tofu. Yet anotheruse of a proteinaceous substance or micro-organism provided by theinvention is the use of bacterial surface display in generatingwhole-cell bioadsorbents or biofilters for environmental purposes,microbiocatalysts, and diagnostic tools

The invention is further explained in the experimental part which cannot be seen as limiting the invention.

EXPERIMENTAL PART Introduction

The major autolysin AcmA of Lactococcus lactis subsp. cremoris MG1363 isan N-acetylmuramidase which is required for cell separation and isresponsible for cell lysis during stationary phase (5, 6). The 40.3-kDasecreted mature protein produces a number of activity bands in azymogram of the supernatant of a lactococcal culture. Bands as small asthat corresponding to a protein of 29 kDa were detected. As no clearingbands are produced by an L. lactis acmA deletion mutant, all bandsrepresent products of AcmA (6). From experimental data and homologystudies we inferred that AcmA likely consists of three domains: a signalsequence followed by an active site domain and a C-terminal regioncontaining three highly homologous repeats of approximately 45 aminoacids which are involved in cell wall binding. As the smallest activeprotein is 29 kDa, it was suggested that the protein undergoesproteolytic breakdown in the C-terminal portion (5, 6).

Cell wall hydrolases of various bacteria and bacteriophages containrepeats similar to those present in AcmA (4, 9, 10, 17). Partiallypurified muramidase-2 of Enterococcus hirae, a protein similar to AcmA,containing 6 similar repeats, binds to peptidoglycan fragments of thestrain (11). The p60 protein of Listeria monocytogenes contains two suchrepeats and was shown to be associated with the cell surface (24).However, which parts of these enzymes contained the binding capacity wasnot assessed in any of these studies.

Nearly all cell wall hydrolases examined so far seem to consist of acatalytic domain and usually, although not always, a domain containing anumber of specific amino acid repeats. In several studies it has beenshown that only a part of some of the cell wall hydrolases is requiredfor enzymatic activity (13, 14, 17, 19, 22, 34). Rashid et al. reportedthe cloning of the gene encoding a 90-kDa glucosamimidase of Bacillussubtilis of which the C-terminus shows significant similarity with theglucosamimidase domain of the S. aureus autolysin (23). The proteincontains two repeated sequences in its N-terminus and two differentrepeats in the middle domain. A deletion derivative lacking theC-terminal 187 amino acids remained tightly bound to the cell walls, butno catalytic activity was observed when expressed in B. subtilis. Bymaking deletions from the N-terminus it was shown that nearly two-thirdsof the protein could be removed without complete loss of cellwall-hydrolyzing activity in E. coli, although loss of more than onerepeat drastically reduced lytic activity.

The N-terminal domain of the major autolysin LytA of Streptococcuspneumonia provides the N-acetylmuramyl-L-alanine amidase catalyticfunction, whereas the C-terminal domain, which contains six repeatedsequences, determines the specificity of binding to the cell wall (forreview: see reference 18). The protein lacks a signal sequence andrequires choline-containing teichoic acids to fully degrade pneumococcalcell walls. Furthermore, it was shown that at least four of the sixrepeats were needed for efficient recognition of the choline residues ofpneumococcal cell walls and the retention of appreciable hydrolyticactivity (7).

LytA, pneumococcal phage lysins as well as clostridial and lactococcalcell wall hydrolases have been used for the construction of activeproteins such that the activity domain and cell wall recognition domainswere exchanged. The N-terminal half of the lactococcal phage enzyme wasfused to the C-terminal domain of LytA (28). The chimeric enzymeexhibited a glycosidase activity capable of hydrolyzingcholine-containing cell walls of S. pneumonia. This result showed thatthe lactococcal phage lysin consisted of at least two domains with aglucosidase activity contained in its N-terminus and two repeats similarto those in AcmA in the C-terminus (6). A tripartite pneumococcalpeptidoglycan hydrolase has been constructed by fusing the N-terminalcatalytic domain of the phage CPL1 lysozyme to HBL3, a protein with anamidase activity and a choline-binding domain (27). The three domainsacquired the proper conformation as the fusion protein behaved as anamidase, a lysozyme and as a choline-dependent enzyme.

Also from nature an enzyme is known having two separate functionalactivity domains: the autolysin gene from Staphylococcus aureus encodesa protein that contains an amidase and an endo-β-N-acetylglucosaminidasedomain separated by three highly similar repeats (20). This protein isprocessed posttranslationally into the two constituting activitydomains.

The aim of the present study was to investigate the modular structure ofAcmA. This was done by consecutively deleting the C-terminal repeats andby fusing the repeats to heterologous proteins. On the basis of cellfractionation and binding studies involving whole cells, it is concludedthat the C-terminal repeats in AcmA bind the autolytic enzyme to thecell wall of L. lactis.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The strains andplasmids used in this study are listed in Table 1. Lactococcus lactiswas grown at 30° C. in two-fold diluted M17 broth (Difco Laboratories,Detroit, Mich.) containing 0.5% glucose and 0.95% β-glycerophosphate(Sigma Chemical Co., St. Louis, Mo.) as standing cultures (½M17). Agarplates of the same medium contained 1.5% agar. Five μg/ml oferythromycin (Boehringer GmbH, Mannheim, Germany) was added when needed.Escherichia coli was grown at 37° C. with vigorous agitation in TYmedium (Difco), or on TY medium solidified with 1.5% agar. Whenrequired, the media contained 100 μg of ampicillin (Sigma), 100 μgerythromycin or 50 μg kanamycin (both from Boehringer) per ml.Isopropyl-β-D-thiogalactopyranoside (IPTG) and5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) (both fromSigma) were used at concentrations of 1 mM and 0.002%, respectively.

General DNA Techniques and Transformation.

Molecular cloning techniques were performed essentially as described bySambrook et al. (25). Restriction enzymes, Klenow enzyme and T4 DNAligase were obtained from Boehringer and were used according to theinstructions of the supplier. Deoxynucleotides were obtained fromPharmacia (Pharmacia Biotech, Uppsala, Sweden). All chemicals used wereof analytical grade and were from Merck (Darmstadt, Germany) or BDH(Poole, United Kingdom). Electrotransformation of E. coli and L. lactiswas performed by using a gene pulser (Bio-Rad Laboratories, Richmond,Calif.), as described by Zabarovsky and Winberg (37) and Leenhouts andVenema (16), respectively. Plasmid DNA was isolated using the QIAGENplasmid DNA isolation kit (QIAGEN GmbH, Hilden, Germany) or byCsCl-ethidiumbromide density gradient centrifugation and DNA fragmentswere isolated from agarose gels using the QIAGEN gel extraction kit andprotocols from QIAGEN.

Primer Synthesis, PCR and DNA Sequencing.

Synthetic oligo deoxyribonucleotides were synthesized with an AppliedBiosystems 392 DNA/RNA synthesizer (Applied Biosystems Inc., FosterCity, Calif.). The sequences of the oligonucleotides used are listed inTable 2.

Polymerase chain reactions (PCR) were performed in a Bio-Medthermocycler 60 (Bio-Med GmbH, Theres, Germany) using super Taq DNApolymerase and the instructions of the manufacturer (HT BiotechnologyLtd., Cambridge, United Kingdom). PCR fragments were purified using thenucleotide removal kit and protocol of QIAGEN.

Nucleotide sequences of double-stranded plasmid templates weredetermined using the dideoxy chain termination method (26) with the T7sequencing kit and protocol (Pharmacia) or the automated fluorescent DNAsequencer 725 of Vistra Systems (Amersham Life Science Inc.,Buckinghamshire, United Kingdom).

Nucleotide and amino acid sequences were analyzed with the PC/GENEsequence analysis program (version 6.8. IntelliGenetics, Inc., Geneva,Switzerland). Protein homology searches in the SWISSPROT, PIR, andGenbank (release Sep. 23, 1996) databases were carried out with theBLAST program (1).

Construction of Acmh Derivatives.

A stop codon and EcoRI restriction enzyme site were introduced in acmAat the end of nucleotide sequences encoding the repeats and at the endof the sequence specifying the 35 active site domain by PCR using theprimers. REPDEL-1 (SEQ ID NO. 1), REPDEL-2 (SEQ ID NO. 2), and REPDEL-3(SEQ ID NO. 3) and plasmid pAL01 as a template. Primer ALA-4 (SEQ ID NO.4), annealing within the sequence encoding the signal peptide of AcmA,was used in all cases as the upstream primer. All three PCR productswere digested with SacI and EcoRI and cloned into 5 the correspondingsites of pBluescript SK+ leading to pDEL1, pDEL2 and pDEL3.Subsequently; the 1,187-bp PfImI-EcoRI fragment of pGKAL1 (5) wasreplaced by the 513, 282. and 76—by Pf1mI-EcoRI fragments of the insertsof pDEL1, 2 and 3, respectively. The proper plasmids specifying proteinscontaining one, two or all three repeats (pGKAL5, 4, and ˜3,respectively) were obtained in L. lactis MG1363acmAΔ1. pGKAL1 was cutwith SpeI. The sticky ends were flushed with Klenow enzyme andself-ligation introduced a UAG stop codon after the Ser 339 codon ofacmA. The resulting plamid was named pGKAL6.

A DNA fragment encoding half of the first repeat until the SpeI site inthe middle of the second repeat was synthesized by PCR using the primersREP-4 A (SEQ ID NO. 5) and B (SEQ ID NO. 6). The NheI and SpeI sites atthe ends of the 250-bp PCR product were cut and the fragment was clonedinto the unique SpeI site of pGKAL1 resulting in plasmid pGKAL7.

Overexpression and Isolation of the AcmA Active Site Domain.

A DNA fragment encoding the active site domain of AcmA was obtainedusing the primers ACMHIS (SEQ ID NO. 7) and REPDEL-3 with plasmid pAL01as a template. The 504-bp PCR fragment was digested with Bg1II and EcoRIand subcloned into the BamHI and BcoRI sites of pET32A (Novagen R&Dsystems Europe Ltd, Abingdon, United Kingdom). The proper construct,pETAcmA, was obtained in E. coli BL21(DE3) (30). Expression of thethioredoxin/AcmA fusion protein was induced in this strain by addingIPTG (to 1 mM final concentration) at an OD600 of 0.7. Four hours afterinduction the cells from 1 ml of culture were collected bycentrifugation and the fusion protein was purified over a Talon™ metalaffinity resin (Clontech Laboratories Inc., Palo Alto, Calif.) using 8 Mureum-elution buffer and the protocol of the supplier. The eluate (200μl) was dialyzed against a solution containing 50 mM NaCl and 20 mM Tris(pH 7) after which CaCl₂ was added to a final concentration of 2 mM. Oneunit of enterokinase (Novagen) was added and the mixture was incubatedat room temperature for 20 h. The protein mixture was dialyzed againstseveral changes of demineralized water before SDS-PAGE analysis and cellbinding studies.

Construction of (3-lactamase and a-amylase Fusions to the AcmA RepeatDomain.

For the introduction of a unique NdeI site at the position of the stopcodon of the E. coli TEM-p-lactamase, the oligonucleotides BETA-1 (SEQID NO. 8) and BETA-2 (SEQ ID NO. 9) were used in a PCR with plasmidpGBL1 (21) as a template. The 403-bp PCR fragment was cut with NdeI andPstI and cloned as a 311-bp fragment into the same sites of pUK21. Theresulting plasmid, pUKblac, was digested with NdeI, treated with Klenowenzyme and subsequently digested with XbaI. The β-lactamase encodingfragment was ligated to an 1,104-bp PvuII-XbaI DNA fragment from pAL01containing the acmA part encoding the repeat region of AcmA. Theresulting plasmid, pUKblacrep, was digested with PstI and DraI and the1349-bp fragment was inserted into the PstI-SnaBI sites of pGBL1,leading to plasmid pGBLR. After digestion of pGAL9 (21) with C1aI andHindIII the 1,049-bp fragment encompassing the 3′-end of the Bacilluslicheniformis α-amylase gene was subcloned into corresponding sites ofpUK21. According to the paper of Perez Martinez et al. (21), thisfragment should be 1,402-bp, but after restriction enzyme analysis itturned out to be approximately 350—by smaller. The resulting plasmid wascalled pUKAL1. A unique EcoRV restriction enzyme site was introduced byPCR at the position of the stop codon of the B. licheniformis α-amylasegene using the oligonucleotides ALFA-A (SEQ ID NO. 10) and ALFA-B (SEQID NO. 11) with plasmid pGAL9 as a template. After restriction of the514-bp PCR fragment with SalI and BcoRV the 440-bp fragment was clonedinto the same sites of pUKAL1 resulting in plasmid pUKAL2. The EcoRV andXbaI sites of this plasmid were used to clone the 1,104-by PvuII-XbaIfragment of pAL01 encoding the repeats of AcmA. The 1,915-bpC1aI-HindIII fragment of the resulting plasmid pUKALR was used toreplace the corresponding 1,049-bp fragment of pGAL9 (pGALR). Allcloning steps described above were performed in E. coli NM522. Theplasmids pGBL1, pGBLR, pGAL9 and pGALR were used to transform L. lactisMG1363 and MG1363acmAΔ1.

SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) and Detection of AcmAand α-amylase Activity.

Two ml of end exponential phase L. lactis cultures were subjected tocentrifugation. 0.5 ml of the supernatant fractions were dialyzedagainst several changes of demineralized water, lyophilized, anddissolved in 0.25 ml of denaturation buffer (3). Cell pellets werewashed with 2 ml of fresh ½M17 medium and resuspended in 1 ml ofdenaturation buffer. Cell extracts were prepared as described by van deGuchte et al. (32).

AcmA activity was detected by a zymogram staining technique usingSDS-PAA (12.5% or 17.5%) gels containing 0.15% autoclaved, lyophilizedMicrococcus lysodeikticus ATCC 4698 cells (Sigma) as described before(6). For the analysis of α-amylase activity 1% starch was included into12.5% PAA gels. After electrophoresis proteins were renatured using theAcmA renaturation solution (3) and the gel was stained with an I₂/KIsolution (at final concentrations of 12 and 18 mM, respectively) (33).

SDS-PAGE was carried out according to Laemmli (15) with the Protean IIMinigel System (Bio-Rad) and gels were stained with Coomassie brilliantblue (Bio-Rad). The standard low range and prestained low and high rangeSDS-PAGE molecular weight markers of Bio-Rad were used as references.

Fractionation of mid- and end-exponential phase cultures of L. lactiswas performed according to the protocol of Baankreis (2).

Binding of AcmA and its Derivatives to Lactococcal Cells.

The cells of 2 ml of exponential phase cultures of MG1363acmAΔ1 weregently resuspended in an equal volume of supernatant of similarly grownMG1363acmAΔ1 carrying either plasmid pGK13, pGKAL1, -3, -4, -5, -6 or -7and incubated at 30° C. for 20 min. Subsequently, the mixtures werecentrifugated. The cell pellets were washed with 2 ml of ½M17 and cellextracts were prepared in 1 ml of denaturation buffer as describedabove, while 0.4 ml of the supernatants were dialyzed againstdemineralized water, lyophilized and dissolved in 0.2 ml of denaturationbuffer.

To analyze competitive binding between AcmA derivatives containing 1 or2 repeats, equal volumes of the supernatants of MG1363acmAΔ1 containingpGKAL3 or pGKAL4 were mixed prior to incubation with the MG1363acmAΔ1cells. The samples were treated for SDS-PAGE as described above.

Three 500 μl samples of a mid-exponential phase culture of MG1363acmAΔ1were centrifugated. From one sample 50 μl of the supernatant werereplaced by 50 μl of a solution containing the AcmA active site domain(see above). 100 μl of the supernatant of sample two were replaced by 50μl demineralized water and 50 μl of the supernatant of a mid-exponentialphase culture of MG1363acmAΔ1 (pGKAL4). Of the third sample 100 μl ofthe supernatant were replaced by 50 μl of the solution containing theAcmA active site domain and 50 μl of the supernatant of MG1363acmAΔ1(pGKAL4). Subsequently the three samples were vortexed to resuspend thecells and incubated for 15 min at 30° C. After centrifugation cell andsupernatant fractions were prepared in 500 μl of denaturation buffer foranalysis of AcmA activity in SDS-(17.5%)PAGE as described above.

Binding of the β-lactamase/AcmA fusion protein was studied by growingMG1363acmAΔ1 containing pGK13, pGBL1 or pGBLR until mid-exponentialphase. The cells of 1 ml of MG1363acmAΔ1(pGK13) culture were resuspendedin an equal volume of supernatant of either of the other two cultures.The mixtures were prepared in duplo and one series was incubated at 30°C. for 5 min while the other was kept at that temperature for 15 min.Then, cell and supernatant fractions were treated as described for theAcmA binding studies, resuspended in denaturaion buffer in half of theoriginal volume, and subjected to SDS-(12.5%)PAGE followed by Westernblot analysis.

Western Blotting and Immunodetection.

Proteins were transferred from SDS-PAA gels to BA85 nitrocellulosemembranes (Schleicher and Schuell, Dassel, Germany) as described before(31). α-amylase and β-lactamase antigen was detected with 2000-folddiluted rabbit polyclonal anti-ampicillinase antibodies (5 prime→+3prime, Inc., Boulder, Co.), and alkaline phosphatase-conjugated goatanti-rabbit antibodies (Promega Corporation, Madison, Wis.) using theWestern-Light Chemiluminescent detection system and protocol (TROPIXInc., Bedford, Mass.).

Enzyme Assays and Optical Density Measurements.

AcmA activity was visualized on ½M17 agar plates containing 0.2%autoclaved lyophilized M. lysodeikticus cells as halo's around coloniesafter overnight growth at 30° C.

α-amylase activity was detected by spotting 10 μl of an overnightculture onto a ½M17 agar plate containing 1% of starch (Sigma). After 18h of incubation at 30° C. halo's were visualized by staining with aniodine solution according to the protocol of Smith et al. (29). Asimilar method was used for the detection of β-lactamase activity (29).

X-prolyl dipeptidyl aminopeptidase (PepX) was measured using thechromogenic substrate Ala-Pro-p-nitroanilid (BACHEM Feinchemicalien AG,Bubendorf, Switserland). After 2 min of centrifugation in an eppendorfmicrocentrifuge 75 μl of a culture supernatant was added to 50 μlsubstrate (2 mM) and 75 μl Hepes buffer (pH 7). The mixture was pipettedinto a microtiter plate well and colour development was monitored in aTHERMOmax microtiter plate reader (Molecular Devices Corporation, MenloOaks, Calif.) at 405 nm during 20 minutes at 37° C. Optical densitieswere measured in a Novaspec II spectrophotometer (Pharmacia) at 600 nm.

RESULTS

Two of the Three Repeats in AcmA are Sufficient for Autolysis and CellSeparation.

Several mutant AcmA derivatives were constructed to investigate thefunction of the three repeats in the C-terminus of AcmA. A stop codonwas introduced behind the codon for Thr-287 (pGKAL4) or Ser-363 (pGKAL3)(see FIG. 1). Plasmid pGKAL4-specified AcmA (A1) only contains the first(most N-terminal) of the three repeats, while pGKAL3 specifies an AcmAvariant (A2) carrying the first two repeats. pGKAL5 specifies an AcmAderivative lacking repeats (A0) due to the introduction of a stop codonafter Ser-218. AcmA specified by pGKAL6 contains one an a half repeat(A1.5) due to the presence of a stop codon behind the Ser-339 codon.From pGKAL7 an AcmA mutant (A4) is produced which carries an additional(fourth) repeat as the result of duplication of the polypeptide fromSer-263 to Thr-338. All proteins were expressed from the acmA promoterin the AcmA-negative strain L. lactis MG1363acmAΔ1. The variousdeletions of AcmA were examined with respect to the followingproperties: (I) their effect on halo formation on plates containing cellwall fragments of M. lysodeikticus, (II) chain length of the cellsexpressing the mutant AcmA's, and sedimentation of the cells in astanding culture, (III) their enzymatic activity, both in the cell andsupernatant fraction and (IV) autolysis.

Halo formation. On a ½M17 plate containing cell wall fragments of M.lysodeikticus halo's were absent when MG1363acmAΔ1 carried pGK13 orpGKAL5. All other strains produced a clear halo that differed in size.The halo size was clearly correlated with the number of full lengthrepeats present, although the addition of an extra repeat resulted in areduced halo size (see Table 3). Apparently, for optimal cell wall lyticactivity a full complement of repeats is required.

Cell separation and sedimentation. The deletion of one and a half, twoand all three repeats had a clear effect on the chain length and onsedimentation of the cells after overnight growth (see Table 3). Thus,efficient cell separation requires the presence of at least two repeatsin AcmA.

Enzyme activity. Cells and supernatants of overnight cultures of allstrains were analyzed for AcmA activity by SDS-PAGE. In the cellfractions no activity was detected for A0, not even after one week ofrenaturation of the protein (Table 3). Of the other derivatives, twomajor activity bands were present in this fraction. In each case theirpositions in the gel corresponded to proteins with the calculatedmolecular weights of the unprocessed and the processed form. (Table 3and not shown). As shown in FIG. 1, all AcmA derivatives were stillactive in the supernatant fractions. AcmA produced the characteristicbreakdown pattern as determined before (FIG. 1, lanes 1 and 3; (6)). AllAcmA derivatives except A0 and A1 also showed a distinct and highlyreproducible degradation pattern. A4 showed 2 additional breakdownproducts after prolonged renaturation (results not shown). These dataindicate that removal of the repeats does not destroy AcmA activity andsuggests that one repeat is sufficient to keep the enzymecell-associated.

Autolysis. To analyze the effect of the repeats on autolysis duringstationery phase, overnight cultures of all strains were dilutedhundred-fold and incubated at 30° C. for 6 days and the decrease ofoptical density (OD₆₀₀) was followed. All cultures exhibited similargrowth rates, reached the same maximal optical densities and did notlyse during the exponential phase of growth. After approximately 60 h ofincubation maximal reduction in OD₆₀₀ was reached in all cases. Theresults are presented in Table 3 and show that the reduction in OD₆₀₀ iscorrelated with the reduction of the number of AcmA repeats. Toinvestigate whether the decrease in OD₆₀₀ really reflected autolysis,the activity of the intracellular enzyme PepX was measured. After 60 hof incubation, PepX activity in the culture medium was also maximal inall samples, decreasing in all cases upon further incubation. Hardly anyPepX activity was detected in the supernatant of the acmAΔ1 mutant andin cultures producing A0, A1 or A1.5. In contrast, a considerablequantity of PepX had released into the supernatant of cultures producingA2 and A3. Thus two repeats in AcmA are sufficient for autolysis of L.lactis. A2 or A4 production led to reduced lysis of the producer cells.Taken together, these results indicate that the repeats in AcmA functionin efficient autolysis and are required for cell separation.

The Active Site Domain of AcmA Resides in the N-Terminal Part.

To examine whether the active site is located in the N-terminal domainof AcmA, a DNA fragment starting at codon 58 until codon 218 of acmA wassynthesized by PCR and fused to the thioredoxin gene in plasmid pET32A.The fusion protein comprises 326 amino acids. A protein with theexpected molecular mass (35 kDa) was isolated from a culture of E. coliBL21(DE3) (pETAcmA) (FIG. 2, lane A2). By cleavage with enterokinase,the protein was split into a thioredoxin part of 17 kDa and an AcmAdomain (nA) of 18 kDa (FIG. 2, lane A1). The zymogram (FIG. 2.B) showsthat the fusion protein did not have appreciable cell wall hydrolyticactivity, while the released domain of AcmA was active (FIG. 2, lanes B1and B2), indicating that the active site domain was in the N-terminalpart of AcmA.

Fusion of the Repeats of AcmA to α-amylase and β-lactamase Yields ActiveEnzymes.

The three C-terminal repeats of AcmA (cA) were fused C-terminally to B.licheniformis α-amylase and E. coli TEM β-lactamase as described inMaterial and Methods and shown in FIG. 3. The hybrid proteins were fusedto the lactococcal signal sequences AL9 and BL1, respectively (21). Bothfusion proteins were active in plate assays, as is only shown for theβ-lactamase/AcmA fusion protein (βcA) (FIG. 4). The halo's aroundcolonies producing the fusion proteins were smaller than those producedby the wild-type enzymes, which could either be caused by reducedintrinsic enzyme activities due to the presence of repeats or byincreased susceptibility to proteolytic degradation. However, thesmaller halo's produced by the chimeric proteins might also be caused byhampered diffusion due to cell wall binding (see below).

The activities of α-amylase and the αcA fusion protein were alsodetected in a renaturing SDS-(12.5%)PAA gel containing 1% starch. Theprimary translation product of the α-amylase gene is a protein of 522amino acid residues which contains a signal sequence of 37 amino acids(21). It is secreted as a 55-kDa protein. αcA consists of 741 aminoacids and, if processed and secreted, would give rise to a 78-kDaprotein. Cell and supernatant fractions of L. lactis MG1363 andMG1363acmAΔ1 carrying pGAL9 or pGALR were analyzed after overnightgrowth of the strains. The results are presented in FIG. 5 and show thatthe clearing bands are present at the position expected for both matureproteins. Apparently, αcA is active. Clearly, smaller products arepresent in the supernatants of the cells producing the fusion protein,the smallest being approximately of the size of wild-type matureα-amylase (FIG. 5 and not shown).The β-lactamase Fusion Protein is Predominantly Present in the CellWall.

To examine whether the presence of the C-terminal domain of AcmAresulted in binding of βcA to the cell wall, mid-exponential phasecultures of L. lactis MG1363acmAΔ1 containing pGBL1, encodingβ-lactamase, or PGBLR, specifying βcA, were fractionated and subjectedto Westen blot analysis (FIG. 6). From pGBL1, β-lactamase is expressedas a protein of 322 amino acids containing a signal sequence of 47 aminoacids. The secreted protein is 30 kDa. βcA consists of 540 amino acidsand is secreted as a protein with a molecular mass of 52 kDa. FIG. 6shows that most of the wild-type β-lactamase is present in the culturesupernatant and none in the cytoplasm. Slightly larger bands, likelyrepresenting the unprocessed form, are found in the membrane fractionsof this strain. In contrast, βcA is predominantly retained in the cellwall fraction, although a considerable amount resides in the cytoplasm,strongly suggesting that the AcmA repeats anchored the hybrid enzyme tothe cell wall. The smaller band present in both cytoplasmic fractions iscaused by cross hybridization of the antibodies to an unspecifiedlactococcal protein (unpublished observation). In the supernatantfraction of cells producing βcA, only little full length protein wasobserved. Several distinct smaller products are present in this fractionwhich were also detectable in very low amounts in the cell wall fractionafter prolonged exposure of the film (not shown) but were absent fromthe other fractions.

The C-terminal Repeats in AcmA are Required for Cell Wall Binding.

Although the results presented in the previous section strongly suggeststhat the C-terminal repeats are required for the retention of protein inthe cell wall, definite proof was obtained by mixing the supernatantfractions of end-exponential phase cultures containing AcmA, or one ofits deletion derivatives (see FIG. 1), with the cells from an equalvolume of a culture of MG1363acmAΔ1 (pGK13). After incubation, cell andsupernatant fractions were examined for the presence of AcmA. Except forA0, all proteins were capable of binding to the MG1363acmAΔ1 cells(Table 3). Also, all degradation products of AcmA and its derivativeswere capable of binding. The finding that A0 was unable to bind wascorroborated by adding the mixture of enterokinase-released nA andthioredoxin to supernatant containing A1. When incubated with AcmA-minuscells, only A1 bound to the lactococcal cells (FIG. 7) as only thisprotein was detectable in the cell fraction. nA was only detected in thesupernatant. This was also the case when the experiment was repeatedwith nA alone (not shown).

Binding of AcmA or βcA to Lactococcal Cells at Different pHs.

The supernatant fraction of a mid-exponential phase L. lactisMG1363acmAΔ1 culture was replaced by the supernatant of amid-exponential phase L. lactis MG1363 culture. This mixture wasincubated at 30° C. for 5 min. Thereafter the supernatant was removed bycentrifugation and the cell pellet was washed with M17. The cell pelletswere dissolved in M17 with pHs ranging from 2 to 10 and incubated at 30°C. for 30 min. The cell and supernatant fractions were separated andtreated as described before and analysed for the presence of AcmAactivity. A similar experiment was executed with mid-exponential phaseL. lactis MG1363acmAΔ1 cells with the supernatant of an L. lactisMG1363acmAΔ1(pGBLR) culture. The presence of βcA was analysed by westernblotting and immunodetection as described.

At all different pHs, AcmA and βcA was found to be bound to thelactococcal cells. The binding of both AcmA and βcA was better at low pHas judged from the activity in a zymogram and the visual presence of theamount of βcA fusion protein in the cell extracts after immunodetection.

Proteolytic Breakdown of AcmA by Pronase and Trypsin.

The supernatant fraction of a mid-exponential phase MG1363acmAΔ1 culturewas replaced by the supernatant of a mid-exponential phase MG1363culture. This mixture was incubated at 30° C. for 15 min. Thereafter thesupernatant and the cell fractions were separated and the cell pelletwas dissolved in an identical volume of M17. To both fractions Pronaseand Trypsin (1 mg/ml) dissolved in 10 mM NaPi buffer (pH=7) was added toan end concentration of (10 μg/ml) and the mixtures were incubated at30° C. Samples were taken after 5 and 30 min and 2 h of incubation. Thecell and supernatant fractions of each sample were separated andprepared for zymographic analysis as described above.

A complete hydrolysis of AcmA by pronase was observed in the supernatantfraction after 2 h of incubation while activity was still present in thecell extract at this time point. The hydrolysis of AcmA by trypsin wasslower and activity was still present in the supernatant after 2 h ofincubation. In time the portion of activity present in the cell extractswas always higher than that observed in the supernatant. These resultsindicate that the AcmA protein is protected when it is bound to thecell.

Binding of AcmA to Different Types of Bacterial Cells.

The strains Bacillus subtilis DB104, Lactobacillus plantarum 80,Streptococcus faecalis JH2-2, Streptococcus thermophilus ATCC 19258,Listeria P, Lactobacillus buchneeri L4, Clostridium beijerinckii CNRZ530 and Escherichia coli NM522 were grown overnight in GM17. Twofractions of each overnight culture were centrifuged and thesupernatants were replaced by the supernatant of an overnight-culture ofL. lactis MG1363acmAΔ1 (pGKAL1) or MG1363acmAΔ1(pGK13). The mixtureswere incubated at 30° C. for 15 min. Subsequently the cell andsupernatant fractions were separated and the cells were washed once withM17 and were prepared for SDS-PAGE as described before and analysed forAcmA activity.

In all cell extracts AcmA activity was present while such an activitywas absent in extracts of cells which had been incubated with thesupernatant of MG1363acmAΔ1(pGK13) which lacks the presence of AcmA.

To investigate the effect of repeat number on binding, equal volumes ofthe supernatants of cultures of MG1363acmAΔ1 (pGKAL3, encoding A2) andMG1363acmAΔ1 (pGKAL4, specifying A1) were mixed. The undiluted and a10-fold diluted mixture were incubated with the AcmA-free cells.Analysis of zymograms of serial dilutions showed that the two activitieswere equally distributed over the cell and supernatant fractions,indicating that both proteins bind equally well (results not shown).

To examine whether the C-terminal repeat sequences of AcmA had thecapacity to bind a heterologous, extracellular enzyme to lactococcalcells, binding of βcA was assessed by incubation of AcmA-minus L. lactiscells with culture supernatants containing either secreted wild-typeβ-lactamase or βcA. As FIG. 8 shows, wild-type β-lactamase wasexclusively present in the supernatant fraction, whereas βcAfractionated with the lactococcal cells and, thus, had bound to thesecells.

DISCUSSION

The results presented in this work indicate that the mature form of theN-acetylmuramidase AcmA of L. lactisconsists of two separate domains.The overproduced and purified N-terminus, from amino acid residue 58 to218 in the pre-protein, is active on M. lysodeikticus cell walls and,thus, contains the active site of the enzyme. This is in agreement withthe finding that the repeat-less AcmA mutant A0 can still hydrolyze M.lysodeikticus cell walls, albeit with severely reduced efficiency.Prolonged renaturation was needed to detect the activity of the enzymein vitro while colonies producing the protein did not form a halo.Enzymes A1 and A2 had in vitro activities which were nearly the same asthat of the wild-type protein, although in the plate assay A1 produced asmaller halo than A2 which, in turn, was smaller than the wild-typehalo. A strain producing A1 grew in longer chains than cells expressingA2 and, in contrast to A2 producing cells, sedimented and did notautolyze. Taken together these results indicate that, although theN-terminus of AcmA contains the active site, the presence of at leastone complete repeat is needed for the enzyme to retain appreciableactivity. Second, only cultures producing AcmA's containing two or morefull length repeats are subject to autolysis and produce wild-type chainlengths. It is tempting to speculate that this apparent increase incatalytic efficiency of AcmA is caused by the repeat domain by allowingthe enzyme to bind to its substrate, the peptidoglycan of the cell wall.As was postulated by Knowles et al. (12) for the cellulase bindingdomains in cellobiohydrolases, such binding would increase the localconcentration of the enzyme. The repeats could be involved in bindingalone or could be important for proper positioning of the catalyticdomain towards its substrate. The increase in AcmA activity with anincreasing number of repeats to up to 3 in the wild-type enzyme,suggests an evolutionary process of repeat amplification to reach anoptimum for proper enzyme-functioning. The binding of A1, A1.5 and A4was comparable with that of wild-type AcmA but these enzyme varietiescaused no or only little autolysis. These observations seem to supportthe idea that 3 repeats are optimal for proper functioning of AcmA. Thepresence of 5 and 6 repeats in the very similar enzymes of E. faecalisand E. hirae, respectively, may reflect slight differences in cell wallstructure and/or the catalytic domain, requiring the recruitment bythese autolysins of extra repeats for optimal enzyme activity.

The hypothesis that the C-terminal domain of AcmA is involved in cellbinding (6) was corroborated in this study. First of all we show thatAcmA is indeed capable of cell binding. AcmA and its derivatives A1,A1.5, A2 and A4 all bound to cells of L. lactis when added from theoutside. To prove that it was the C-terminus of AcmA that facilitatedbinding and not some intrinsic cell wall binding capacity of theN-terminal domain, the repeat domain was fused to two heterologousproteins which do not normally associate with the cell wall. The smallerhalo's produced by αcA and βcA compared to the wild-type proteins andthe presence of most of βcA in the cell wall fraction are indicative ofcell binding of the fusion proteins via the AcmA-specific repeats.

The βcA binding studies clearly show that it is the AcmA repeat domainthat specifies cell wall binding capacity: whereas wild-type β-lactamase(and, for that matter, repeat-less AcmA) did not bind to lactococcalcells, βcA did bind to these cells when added from the outside. Theresults obtained with A1 in the binding assay show that only one repeatis sufficient to allow efficient binding of AcmA. In a separate study(5) we showed that AcmA can operate intercellularly: AcmA-freelactococcal cells can be lyzed when grown together with cells producingAcmA. Combining this observation with the results presented above allowsto conclude that AcmA does not only bind when confronting a cell fromthe outside but, indeed, is capable of hydrolyzing the cell wall withconcomitant lysis of the cell.

AcmA-like repeats were found to be present at different locations inmore than 30 proteins after a comparison of the amino acid sequences ofthe repeats in AcmA with the protein sequences of the Genbank database(release 23). Not all of these proteins with repeats varying from one tosix are cell wall hydrolases. Alignment of the amino acid sequences ofall the repeats yielded a consensus sequence similar to that postulatedby Birkeland and Hourdou et al. (4, 9). Interestingly, if a limitednumber of modifications are allowed in the consensus repeat, the repeatis also present 12 and 4 times, respectively, in two proteins ofCaenorhabditis elegans, which both show homology with endochitinases(Gene accession numbers U64836 and U70858) (36). Possibly, these repeatsanchor these enzymes to fungi ingested by this organism. The presence ofsimilar repeats in proteins of different bacterial species stronglysuggests that they recognize and bind to a general unit of thepeptidoglycan. An interesting goal for the future will be to elucidatethe unit to which they bind and the nature of the binding.

As has been reported earlier for intact AcmA (5), and, as we show herefor its C-terminal deletion derivatives, the enzyme is subject toproteolytic degradation. None of the degradation products were presentin cell extracts of whole cells indicating that they are not formedinside the cell (data not shown). The degradation pattern of each AcmAderivative is specific and very reproducible. Based on the sizes of thedegradation products, a number of the proteolytic cleavage sitesprobably resides in the intervening sequences. One such site (1 inFIG. 1) is present between repeat 1 and 2. Cleavage at this positionwould result in an active protein of approximately 28 kDa, which isindeed seen in the supernatants of all strains producing AcmA with 1.5or more repeats. A second cleavage site is probably located between thesecond and third repeat (2 in FIG. 1). Cleavage at this site is eitherrather infrequent, or the resulting degradation product is not veryactive, which, in both cases, would lead to the faint bands of activityobserved in lanes 1 and 3 of the zymogram presented in FIG. 1. Thepresence of cleavage sites in between the AcmA repeats is furthersuggested by the presence of specific degradation products observed inαcA and μcA; their sizes are in accord with the location of the cleavagesites postulated in AcmA. In addition, as also bands of the size of thewild-type α-amylase and β-lactamase are observed, an additional cleavagesite seems to be present around the fusion point of these enzymes andthe cell wall binding domain of AcmA.

All degradation products of AcmA and those of the two fusion proteinsare mainly present in the supernatant and to some extent in the cellwall fraction, but not in the cells. As none of the L. lactis strainsused produced the cell wall-anchored proteinase PrtP, this enzyme cannot be held responsible for the specific degradation of AcmA or thefusion proteins. Apparently, an extracellular proteinase exists in L.lactis that is capable of removing the repeats, which may represent amechanism for the regulation of AcmA activity.

TABLE 1 Bacterial strains and plasmids used in this study. Source orStrain or plasmid Relevant phenotype(s) or genotype(s) reference StrainsL. lactis subsp. cremoris MG1363 Plasmid-free strain (8) MG1363 acmAΔ1Derivative of MG1363 carrying a 701-bp SacI-SpeI deletion in acmA (6) E.coli NM522 supE thi Δ(lac-proAB) Δhsd5(r_(k) ⁻,m_(k) ⁻) [F′ proABlacI^(q)ZM15] Stratagene BL21 (DE3) ompT r_(B) ⁻m_(B) ⁻ int;bacteriophage DE3 lysogen carrying the T7 RNA (30) polymerase genecontrolled by the lacUV5 promoter Plasmids pET32A Ap^(r), vector forhigh level expression of thioredoxin fusion Novagen proteins pUK21Km^(r), general cloning vector (35) pBluescript SK+ Ap^(r), generalcloning vector Stratagene pAL01 Ap^(r), pUC19 carrying a 4,137-bplactococcal chromosomal DNA insert (6) with acmA gene pDEL1 Ap^(r),pBluescript SK+ with 785-bp SacI-EcoRI fragment of acmA This workobtained by PCR with primers ALA-4 and REPDEL-1 pDEL2 Ap^(r),pBluescript SK+ with 554-bp SacI-EcoRI fragment of acmA This workobtained by PCR with primers ALA-4 and REPDEL-2 pDEL3 Ap^(r),pBluescript SK+ with 348-bp SacI-EcoRI fragment of acmA This workobtained by PCR with primers ALA-4 and REPDEL-3 pGKAL1 Em^(r), Cm^(r),pGK13 containing acmA under control of its own promoter (5) on a1,942-bp SspI-BamHI insert pGKAL3 Em^(r), Cm^(r), pGKAL1 derivativeexpressing A2 This work pGKAL4 Em^(r), Cm^(r), pGKAL1 derivativeexpressing A1 This work pGKAL5 Em^(r), Cm^(r), pGKAL1 derivativeexpressing A0 This work pGKAL6 Em^(r), Cm^(r), pGKAL1 derivativeexpressing A1.5 This work pGKAL7 Em^(r), Cm^(r), pGKAL1 derivativeexpressing A4 This work pETAcmA Ap^(r), pET32A expressing active sitedomain of AcmA from residues 58 This work to 218 fused to thioredoxinpGBL1 Em^(r), pWV01 derivative expressing E. coli TEM-β-lactamase fusedto (21) export element BL1 of L. lactis pGAL9 Em^(r), pWV01 derivativeexpressing B. licheniformis α-amylase fused (21) to export element AL9of L. lactis pUKAL1 Km^(r), pUK21 with ±1,050-bp ClaI-HindIII fragmentof pGAL9 This work pUKAL2 Km^(r), pUKAL1 in which the ±650-bp SalI-EcoRVfragment is replaced This work by the 440-bp SalI-EcoRV fragment of thePCR fragment obtained with primers ALFA-A and -B pUKALR Km^(r), pUKAL2with 1,104-bp PvuII-XbaI fragment of pAL01 in EcoRV This work and XbaIsites pUKblac Km^(r), pUK21 with 311-bp PstI-NdeI PCR fragment obtainedwith This work primers BETA-1 and -2 pUKblacR Km^(r), pUKblac carrying1,104-bp PvuII-XbaI fragment of pAL01 in This work NdeI and XbaI sitespGBLR Em^(r), pGBL1 expressing the β-lactamase/AcmA fusion protein Thiswork pGALR Em^(r), pGAL9 expressing the α-amylase/AcmA fusion proteinThis work

R/E Name Nucleotide Sequence (5′→3′) Site REPDEL-CGCGAATTCAGATTATGAAACAATAAG SEQ ID NO: 1 EcoRI 1 REPDEL-CGCGAATTCTTATGTCAGTACAAGTTTTTG SEQ ID NO: 2 EcoRI 2 REPDEL-CGCGAATTCCTTATGAAGAAGCTCCGTC SEQ ID NO: 3 EcoRI 3 ALA-4CTTCAACAGACAAGTCC SEQ ID NO: 4 REP-4A AGCAATACTAGTTTTATA SEQ ID NO: 5SpeI REP-4B CGCGAATTCGCTAGCGTCGCTCAAATTCAAAGTGCG NheI SEQ ID NO: 6ACMHIS AGGAGATCTGCGACTAACTCATCAGAGG SEQ ID NO: 7 BglII BETA-1GGATCATGTAACTCGCC SEQ ID NO: 8 BETA-2 GGAATTCCATATGCTTAATCAGTGAGG SEQ IDNO: 9 NdeI ALFA-A GCATCCGTTGAAAGCGG SEQ ID NO: 10 ALFA-BGAATTCGATATCTTTGAACATAAATTG SEQ ID NO: 11 EcoRV ALA-14 GATAAATGATTCCAAGCSEQ ID NO: 12 ALA-22 CTCAAATTCAAAGTGCG SEQ ID NO: 13

TABLE 3 Properties of L. lactis expressing AcmA derivatives. AcmA StrainAcmA % Reduction PepX Chain activity^(i)) Cell Number^(a))(plasmid)^(b)) variant^(c)) in OD₆₀₀ ^(d)) activity^(e)) length^(f))Halo^(g)) Sedimentation^(h)) sup ee binding^(j)) 1 MGpGK13 A3 32.6 16.9A 3.1 − + + + 2 Δ1pGK13 — 15.2 0.3 C 0 + − − − 3 Δ1pGKAL1 A3 36.7 19.8 A5.0 − + + + 4 Δ1pGKAL3 A2 29.3 13.3 A 4.6 − + + + 5 Δ1pGKAL4 A1 18.8 0.4B 3.9 + + + + 6 Δ1pGKAL5 A0 15.6 0.3 C 0 + + − − 7 Δ1pGKAL6 A1.5 18.61.6 B 2.2 ± + + + 8 Δ1pGKAL7 A4 21.1 4.9 A 4.0 − + + + ^(a))The numbercorresponds to the AcmA derivative produced, as schematized in FIG. 1.^(b))MG: L. lactis MG1363, Δ1: L. lactis MG1363acmAΔ1. ^(c))—: no AcmAproduced; Ax: AcmA with x repeats. ^(d))The OD₆₀₀ reduction wascalculated using the following formula: [(OD_(max.) −OD_(60 hours))/OD_(max.)] * 100%. ^(e))Activity is in arbitrary unitsmeasured as the increase of absorption at 405 nm in time. ^(f))Endexponential phase ½M17 cultures were subjected to light microscopicanalysis. A: mainly single cells and some chains up to 5 cells B: somesingle cells but mainly chains longer than 5 cells C: no single cells,only very long chains ^(g))The sizes of the halo's were measured inmillimeters from the border of the colony after 45 h of incubation at30° C. ^(h))Analyzed by visual inspection of standing 1/2 M17 culturesafter overnight growth in test tubes. ^(i))Judged from zymograms ofsamples from end-exponential phase ½M17 cultures; sup: supernatantfraction, ce: cell-extract. ^(j))Binding of AcmA derivatives insupernatants of end-exponential phase 1/2 M17 cultures toend-exponential phase cells of L. lactis MG1363acmAΔ1 after 20 min ofincubation at 30° C. (see text for details).

FIG. 1. Analysis of AcmA activity in supernatant fractions ofend-exponential-phase cultures of MG1363 containing pGK13 (1) andMG1363acmAΔ1 containing either pGK13, not encoding AcmA (2), pGKAL1,encoding enzyme A3 (3), pGKAL3, encoding enzyme A2 (4), pGKAL4, encodingenzyme A1 (5), pGKAL5, encoding enzyme A0 (6), pGKAL6, encoding enzymeA1.5 (7), or pGKAL7, encoding enzyme A4 (8) in a renaturingSDS-(12.5%)PAA gel containing 0.15% M. lysodeikticus autoclaved cells.Molecular masses (in kilodaltons, kDa) of standard proteins (lane M) areshown in the left margin. Below the gel the lower part of lanes 5, 6 and7 of the same gel is shown after one week of renaturation. The righthalf of the figure gives a schematic representation of the various AcmAderivatives. SS (black), signal sequence; Rx (dark grey), repeats; lightgrey, Thr, Ser and Asn-rich intervening sequences (6); arrows,artificially duplicated region in the AcmA derivative containing fourrepeats. The active site domain is shown in white. MW, expectedmolecular sizes in kDa of the secreted forms of the AcmA derivatives.The numbers of the AcmA derivatives correspond with the lane numbers ofthe gel. Numbered arrowheads indicate the putative location ofproteolytic cleavage sites.

FIG. 2. Purification of the AcmA active site domain (nA). (A)SDS-(12.5%) PAGE of cell extract of 10 μl of E. coli BL21(DE3) (pETAcmA)(lane 3) induced for 4 h with IPTG. Lane 2, 10 μl of purified fusionprotein isolated from 25 μl of induced E. coli culture and lane 1, 10 μlof the enterokinase cleft protein. (B) Renaturing SDS-(12.5%) PAGE with0.15% M. lysodeikticus autoclaved cells using the same amount of thesamples 1 and 2 shown in part A. Molecular masses (in kilodaltons) ofstandard proteins are shown on the left of the gel. Before loading thesamples were mixed with an equal volume of 2× sample buffer (15).

FIG. 3. Schematic representation of plasmids pGBLR and pGALR carrying,respectively, C-terminal fusions of the repeats of AcmA to β-lactamaseand α-amylase. α-amy, α-amylase gene of B. licheniformis; β-lac,β-lactamase gene of E. coli; acmA, 3-prime end of the N-acetylmuramidasegene of L. lactis MG1363 encoding the three repeats; EmR and CmR,erythromycin and chloramphenicol resistance genes; AL9 and BL1, proteinsecretion signals from L. lactis MG1363 (21); repA and ORI, gene for thereplication protein and origin of replication of the lactococcal plasmidpWV01, respectively; Pspo2, B. subtilis phage Spo2 promoter. Black boxesindicate the PCR fragments used for the introduction of the restrictionenzyme sites EcoRV and NdeI at the position of the stopcodons of theα-amylase and β-lactamase genes, respectively. The open box indicatesthe part which has been subcloned into pUK21 for construction work. Thegrey boxes show the fragment of pAL01 used to fuse the 3′-end of acmA tothe α-amylase and β-lactamase genes. Only relevant restriction enzymesites are shown.

FIG. 4. β-lactamase activity in L. lactis. Activity of wild-typeβ-lactamase and its AcmA fusion derivative (βcA) produced by cells of L.lactis MG1363 and MG1363acmAΔ1 containing pGK13, pGBL1 or PGBLR. The½M17 agar plate was stained with iodine after overnight growth of thecolonies according the protocol of Smith et al. (29).

FIG. 5. α-amylase activity in the supernatant of L. lactis. Activity ofwild-type α-amylase (α) and the αcA fusion protein in an SDS-(12.5%)PAAgel containing 1% starch. The proteins were renatured by washing the gelwith Triton X-100 and subsequently stained with iodine (33). Theequivalent of 40 μl of supernatant of ½M17 cultures of L. lactis MG1363(M) and MG1363acmAΔ1 (A) containing pGAL9 or pGALR were loaded onto thegel. Molecular masses (in kDa) of standard proteins are shown in theleft margin.

FIG. 6. Localization of β-lactamase in L. lactis. Western blot analysisof fractions of MG1363acmAΔ1 expressing β-lactamase (from pGBL1) or βcAfusion protein (encoded by PGBLR) using polyclonal antibodies directedagainst β-lactamase. Amount of samples loaded is equal to 200 μl ofculture. Fractions: S, supernatant; CW, cell wall; CY, cytoplasm; MB,membrane-associated; and M, membrane.

FIG. 7. Analysis of the binding of AcmA derivatives nA and A1 by arenaturing SDS-(17.5%) PAGE with 0.15% M. lysodeikticusautoclaved cells.Cell (C) and supernatant (S) fractions of MG1363acmAΔ1 cells incubatedwith nA and A1 from the culture supernatant of MG1363acmAΔ1 containingpGKAL4. 60 μl of the samples were loaded. Molecular masses (in kDa) ofstandard proteins are shown in the left margin.

FIG. 8. Binding of the βcA fusion protein to L. lactis. The figure showsa Western blot using polyclonal antibodies against β-lactamase. Cellextracts (lanes 1, 3, 5) and supernatants (lanes 2, 4, 6) ofmid-exponential phase MG1363acmAΔ1 (pGK13) cells incubated for 5 minuteswith supernatants of MG1363acmAΔ1 containing pGK13 (lanes 1, 2), pGBLR(lanes 3, 4) or pGBL1 (lanes 5, 6), respectively. The positions ofwild-type β-lactamase (β) and the βcA fusion protein are indicated onthe right. Molecular masses (in kDa) of standard proteins are shown inthe left margin. Twenty μl of samples were loaded onto an 12.5% PAA gel.

FIG. 9. Schematic representation of the AcmA protein. SS (black), signalsequence; R (dark grey), repeats; shaded regions, intervening sequences.The active site domain is shown in white.

FIG. 10. Amino acid sequence alignment of the repeats of AcmA in L.lactis plus consensus sequence. Amino acid sequences SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 are depicted.

FIG. 11. Amino acid sequence alignment of repeats in various (SEQ ID NO:20 through SEQ ID NO: 110), wherein:

SEQ ID NO: Species and Protein Residue No. SEQ ID NO: 20 Lactobacilluslactis acmA 245–287 (33) SEQ ID NO: 21 321–363 (31) SEQ ID NO: 22395–437 SEQ ID NO: 23 Enterococcus faecalis autolysin 363–405 (25) SEQID NO: 24 431–473 (25) SEQ ID NO: 25 499–541 (25) SEQ ID NO: 26 567–609(19) SEQ ID NO: 27 629–671 SEQ ID NO: 28 Enterococcus hirae mur2 257–299(38) SEQ ID NO: 29 338–380 (33) SEQ ID NO: 30 414–456 (32) SEQ ID NO: 31489–531 (33) SEQ ID NO: 32 565–607 (15) SEQ ID NO: 33 623–665 SEQ ID NO:34 Lactococcus øTuc2009 lys 332–375 (10) SEQ ID NO: 35 386–428 SEQ IDNO: 36 Lactococcus ø-LC3 lysB 333–376 (10) SEQ ID NO: 37 387–429 SEQ IDNO: 38 Bacillus øPBSX xy1A 161–204 SEQ ID NO: 39 Bacillus ø PZA orf 15163–207 (6) SEQ ID NO: 40 (=ø–29) 214–258 SEQ ID NO: 41 Bacillus ø B103orf 15 165–209 (9) SEQ ID NO: 42 219–263 SEQ ID NO: 43 Bacillus øgle lys399–442 SEQ ID NO: 44 Bacillus sphaericus Pep I  3–46 (6) SEQ ID NO: 45 53–96 SEQ ID NO: 46 Haemophilus influenzae amia 294–336 SEQ ID NO: 47387–430 SEQ ID NO: 48 Listeria monocytogenes P60  30–72 (130) SEQ ID NO:49 203–245 SEQ ID NO: 50 Listeria innocua P60  30–72 (130) SEQ ID NO: 51201–243 SEQ ID NO: 52 Listeria ivanovii P60  30–72 (125) SEQ ID NO: 53198–240 SEQ ID NO: 54 314–356 SEQ ID NO: 55 Listeria seeligeri P60 30–72 (127) SEQ ID NO: 56 200–242 (75) SEQ ID NO: 57 320–362 SEQ ID NO:58 Listeria welshimeri P60  30–72 (127) SEQ ID NO: 59 198–240 (75) SEQID NO: 60 316–358 SEQ ID NO: 61 Listeria grayi P60  30–72 (104) SEQ IDNO: 62 177–219 (79) SEQ ID NO: 63 299–342 SEQ ID NO: 64 Escherichia coliyebA  77–121 SEQ ID NO: 65 Haemophilus influenzae yebA 131–174 SEQ IDNO: 66 Escherichia coli nlpD 123–166 SEQ ID NO: 67 Haemophilusinfluenzae lppB 147–190 SEQ ID NO: 68 Haemophilus somnus lppB 120–164SEQ ID NO: 69 Pseudomonas aeruginosa lppB  69–113 SEQ ID NO: 70Synechocystis nlpD 187–130 SEQ ID NO: 71 Sinohizobium meliloti nlpD166–209 SEQ ID NO: 72 Escherichia coli dniR 113–155 (16) SEQ ID NO: 73172–213 SEQ ID NO: 74 Staphylococcus aureus ProtA 431–474 SEQ ID NO: 75Bacillus subtilis papQ  28–70 (17) SEQ ID NO: 76  88–130 (20) SEQ ID NO:77 151–193 SEQ ID NO: 78 Bacillus subtilis spoVID 525–568 SEQ ID NO: 79Escherichia coli  50–93 SEQ ID NO: 80 Synechocystis  4–47 SEQ ID NO: 81Bacillus subtilis yaaH  1–43 (5) SEQ ID NO: 82  49–92 SEQ ID NO: 83Bacillus subtilis yhdD  29–71 SEQ ID NO: 84  94–136 (29) SEQ ID NO: 85176–218 (23) SEQ ID NO: 86 242–284 (24) SEQ ID NO: 87 309–353 SEQ ID NO:88 Caenorhabditis elegans  23–66 (11) SEQ ID NO: 89  78–121 (21) SEQ IDNO: 90 143–186 (21) SEQ ID NO: 91 208–251 (19) SEQ ID NO: 92 271–314(20) SEQ ID NO: 93 335–378 (23) SEQ ID NO: 94 402–445 (21) SEQ ID NO: 95467–510 (37) SEQ ID NO: 96 548–591 (44) SEQ ID NO: 97 636–679 (66) SEQID NO: 98 746–786 (8) SEQ ID NO: 99 795–838 SEQ ID NO: 100Caenorhabditis elegans  23–66 (51) SEQ ID NO: 101 118–161 (25) SEQ IDNO: 102 187–226 (9) SEQ ID NO: 103 236–279 SEQ ID NO: 104 Bacillussubtilis 191–136 SEQ ID NO: 105 Citrobacter fruendii eae  65–113 SEQ IDNO: 106 Escherichia coli eae  65–113 SEQ ID NO: 107 Bacillus subtilisyneA  40–90 SEQ ID NO: 108 Streptococcus pyogenes  47–103 SEQ ID NO: 109Bacillus subtilis yqbp 177–234 SEQ ID NO: 110 Bacillus subtilis 161–218a) Proteins listed were obtained by a homology search in the SWISSPROT,PIR, and Genbank databases with the repeats of AcmA using the BLASTprogram (1). b) *; genes encoding cell wall hydrolases. #; proteinscontaining repeats that are longer than average. c) The number of aminoacid residues between the repeats are given between brackets. d) Numberof amino acids of the primary translation product. e) Genbank accessionnumber.

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1. A method for attaching a substance to the cell wall of a microorganism, said method comprising: attaching the substance to a cell wall of the microorganism with an attaching peptide comprising at least one AcmA repeat comprising SEQ ID NO:17, wherein the AcmA repeat is capable of attaching the substance to the cell wall of the microorganism.
 2. The method according to claim 1, wherein the attaching peptide originates from the major peptidoglycan hydrolase of Lactococcus lactis.
 3. The method according to claim 1, further comprising: wherein attaching the substance to the cell wall of the microorganism comprises attaching an antigenic substance to the cell wall of the microorganism; and formulating said microorganism with suitable pharmaceutical excipients, thus producing a vaccine.
 4. The method according to claim 1 further comprising: wherein attaching the substance to the cell wall of the microorganism comprises attaching an antigenic substance to the cell wall of the microorganism; formulating said microorganism with suitable pharmaceutical excipients to form a formulation; and injecting a subject with said formulation.
 5. In a method for attaching a heterologous peptide to a recombinant microorganism's outer surface, an improvement comprising: using a peptide comprising at least one AcmA repeat to attach the heterologous peptide to the outer surface of the recombinant microorganism, wherein, structurally, the at least one AcmA repeat comprises SEQ ID NO:17, and wherein, functionally, the at least one AcmA repeat is able to attach a substance to a cell wall of a microorganism.
 6. A process for anchoring a substance to a microorganism's cell wall, said process comprising: attaching a substance to an AcmA means for binding to the cell wall of the microorganism, wherein said AcmA means for binding to the cell wall of the microorganism comprises the amino acid sequence of SEQ ID NO:17; and placing the AcmA means for binding to the cell wall of the microorganism attached to the substance in contact with a microorganism so that the AcmA means for binding to the cell wall of the microorganism anchors to the microorganism's cell wall.
 7. The method according to claim 1, wherein the microorganism is a non-recombinant microorganism.
 8. The method according to claim 3, wherein the microorganism is a non-recombinant microorganism.
 9. The method according to claim 4, wherein the microorganism is a non-recombinant microorganism.
 10. The method according to claim 1, wherein the substance is selected from the group consisting of an antigenic determinant, an enzyme, an antibody, a single-chain antibody, a fragment of an antibody, a fragment of a single chain antibody, a polyhistidyl tag, a fluorescing protein, luciferase, a binding peptide, an antibiotic, a hormone, a non-peptide antigenic determinant, a carbohydrate, a fatty acid, an aromatic compound, and a reporter molecule.
 11. The method according to claim 3, further comprising injecting a subject with the vaccine that was produced. 